一株关中热泉液口普鲁兰酶产生菌的筛选鉴定及部分酶学性质研究

Identification and evaluation of enzyme activities of pullulanase producing strain WSCF46 isolated from hydrothermal ventin Guanzhong Area

[目的]筛选性能良好的产普鲁兰酶的菌株,对菌株进行多项分类鉴定,初步分离所产生的普鲁兰酶并进行性质研究。[方法]利用糯米粉作唯一碳源分离纯化普鲁兰酶产生菌,通过形态特征观察、生理生化测定、16S r RNA序列分析等实验确定菌株的分类地位。利用硫酸铵沉淀法得到粗酶,分析了酶的最适温度、最适p H、温度和p H稳定性、Na Cl等的耐受性。通过加入普鲁兰多糖诱导物优化培养基,提高普鲁兰酶产量。[结果]从我国陕西地下热泉样品中分离得到的一株普鲁兰酶产生菌WSCF46,经过多项分类鉴定显示其是与Vibrio parahaemolyticus亲缘性最近。菌株WSCF46所产的普鲁兰酶反应最适p H为5.0,最适温度60℃,经诱导培养优化后平均酶活达2.7U/m L。[结论]多项分类结果鉴定菌株为Vibrio parahaemolyticus strain ATCC17802,该菌株产普鲁兰酶相关报道在国内为首次。普鲁兰酶产量较其他来源的酶相比较高,具有进一步研究价值。

[Objective]To isolate and identify an pullulanse producing bacterium, purify and characterize pullulanasepreliminary.[Methods]Pullulanse-producing bacterium WSCF46 was isolated by pullulan from water samples of hydrothermal vent in Dongda village, China. The morphological, biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain WSCF46. Crude pullulanase produced by strain WSCF46 was purified by ammonium sulfate precipitation. The enzyme properties including optimal temperature, optimal pH, thermal stability, pH stability, NaCl tolerance were tested. Pullulan inducerwas added in the medium to improve pullulanase production.[Results]Strain WSCF46 was gramnegative bacterium, its growth pH range was from 2.0 to 8.5 and growth temperature range was from 25℃ to 85℃.Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain WSCF46 was a member of Vibrio. The extracellular pullulanase activity was showed at pH 5.0 and 60℃ . The pullulanase was stable up to 65℃ and retained about 60% residual at 65℃ for 2h. Average enzyme activity reached 2.7u/mL when use pullulan as carbon source inducer. [Conclusion] Polyphasic taxonomy revealed that strain WSCF46 is most closely related to Vibrio parahaemolyticus strain ATCC 17802. This is the first time which pullulanase was producedin China. Strain WSCF4 could be used in further study because of its high production compared with other sources.